Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States of America.

Abstract

In laboratory yeast strains with Sir2 and Fob1 function, wild-type NAD+ salvage is required for calorie restriction (CR) to extend replicative lifespan. CR does not significantly alter steady state levels of intracellular NAD+ metabolites. However, levels of Sir2 and Pnc1, two enzymes that sequentially convert NAD+ to nicotinic acid (NA), are up-regulated during CR. To test whether factors such as NA might be exported by glucose-restricted mother cells to survive later generations, we developed a replicative longevity paradigm in which mother cells are moved after 15 generations on defined media. The experiment reveals that CR mother cells lose the longevity benefit of CR when evacuated from their local environment to fresh CR media. Addition of NA or nicotinamide riboside (NR) allows a moved mother to maintain replicative longevity despite the move. Moreover, conditioned medium from CR-treated cells transmits the longevity benefit of CR to moved mother cells. Evidence suggests the existence of a longevity factor that is dialyzable but is neither NA nor NR, and indicates that Sir2 is not required for the longevity factor to be produced or to act. Data indicate that the benefit of glucose-restriction is transmitted from cell to cell in budding yeast, suggesting that glucose restriction may benefit neighboring cells and not only an individual cell.

(A) RLS analysis for wild-type strain BY4741 in 2% glucose and glucose-restricted conditions indicates that the longevity benefit of CR is lost upon migration to new plate locations. (B) Percent increases in median life span relative to RLS on 2% glucose without migration. Data were collected from four independent experiments with n = 30. *, p < 0.05; ***, p < 0.001 in comparison to the 2% not moved group.

Supplementation with NA restores the longevity benefit to moved glucose-restricted cells.

(A) RLS analysis for wild-type BY4741 in 2% glucose and glucose-restricted conditions indicates that supplementation with NA is sufficient to restore the longevity benefit of CR upon migration to new plate locations. (B) Percent increases in median life span relative to RLS on 2% glucose without migration. Data were collected from four independent experiments with n = 30. *, p < 0.05; **, p < 0.01; *** p, < 0.001 in comparison to the 2% not moved group.

Supplementation with concentrated conditioned media restores the longevity benefit to moved glucose-restricted cells.

(A) RLS analysis for wild-type BY4741 in 2% glucose and glucose-restricted conditions indicates that supplementation with concentrated conditioned media is sufficient to restore the longevity benefit of CR upon migration to new plate locations. (B) Percent increases in median life span relative to RLS on 2% glucose without migration. Data were collected from four independent experiments with n = 30. ***, p < 0.001 in comparison to the 2% not moved group.

A low molecular weight factor in CR-conditioned media is required for the longevity benefit of CR.

(A) RLS analysis for wild-type BY4741 in dialyzed 2% glucose and glucose-restricted conditions indicates that the longevity factor is a small molecule. (B) Percent increases in median life span relative to RLS on 2% glucose without migration. Data were collected from four independent experiments with n = 30. ns, no significant difference; *, p < 0.05; ***, p < 0.001 in comparison to the 2% not moved group.

Sir2 is not required for production or action of the longevity factor.

(A) RLS analysis for sir2 fob1 double mutant yeast in 2% glucose and glucose-restricted conditions indicates that the longevity benefit of CR is lost upon migration to new plate locations without Sir2. (B) Percent increases in median life span relative to RLS on 2% glucose without migration. Data were collected from three independent experiments with n = 60. **, p < 0.01; *** p < 0.001 in comparison to the 2% not moved group. (C) RLS analysis for wild-type BY4741 in 2% glucose and glucose-restricted conditions indicates that supplementation with concentrated conditioned media from sir2 fob1 mutant yeast restores longevity benefit of CR upon migration to new plate locations. (D) Percent increases in median life span relative to RLS on 2% glucose without migration. Data were collected from three independent experiments with n = 45. ***, p < 0.001 in comparison to the 2% not moved group.